Therapeutic human albumin solutions with low prekallikrein activator (PKA) activity and process for obtaining them

ABSTRACT

The invention discloses a purified albumin solution of human origin with low prekallicrein activator (PKA) activity and stability over time characterized in that it has an antithrombin content equal to or greater than 0.03 mg/g of albumin, and a process for production thereof by the partial extraction of the antithrombin during fractionation of the human plasma.

This application is a Continuation of U.S. application Ser. No.11/185,613, filed Jul. 19, 2005 (now U.S. Pat. No. 7,332,577), whichclaims priority under 35 U.S.C. §119(e) to Spanish application SerialNo. 200401830, filed Jul. 26, 2004, both of which are incorporatedherein by reference in their entirety.

DESCRIPTION

The present invention relates generally to therapeutic human albuminsolutions with low prekallikrein activator (PKA) activity, which arestable over time, and also to a process for reducing prekallikreinactivator activity in purified albumin solutions of human origin.

State of the Art

Coagulation factor XII (Hageman factor) is a protein having a molecularweight of approximately 80,000 which, in its activated form, consists offragments thereof having a molecular weight of approximately 28,000.This activated factor XII (fXIIa) acts as a prekallikrein activator(PKA).

Apart from its action on blood coagulation mechanisms, PKA acts onprekallikrein, catalysing its conversion to kallikrein which adverselyaffects the conversion from kininogen to bradykinin. Bradykinin is apotent vasodilator which can cause incidences of hypotension. Thekallikrein formed also catalyses the formation of PKA, feeding back theprocess.

Purified human plasma albumin solutions are therapeutically useful andare widely used to increase blood volume in cardiovascular surgery,among other uses.

However, the hypotensive effect caused by the rapid infusion of humanalbumin solutions is one of the adverse reactions, which is serious inspecific cases and frequently occurs in conjunction with this infusionof albumin.

PKA may be present as a contaminant in these human albumin solutions, asit is generated from factor XII by contact with foreign surfaces duringthe albumin purification process and for other reasons, so its contentin said solutions has been limited to levels of less than 35 IU/ml(European Pharmacopoeia).

In batches of commercial albumin, which have low PKA levels on theproduction date, it has been found that the PKA level increases overtime during the storage thereof within the established period ofstability.

Starting from this state of the art, the inventors proposed to findtherapeutic human albumin solutions having low prekallikrein activator(PKA) activity and also ensuring significant stability of the PKA levelsin the commercial albumin solution for the established storage period sothat a therapeutic human albumin solution with a very low level ofprekallikrein activator is clinically available at any time within apreviously established long storage period.

After carrying out extensive research and investigations, the inventorshave found that it is possible to anticipate the generation of PKAactivity in a human albumin solution and simultaneously to obtain a highdegree of stability over time by setting limits to the quantity ofantithrombin in the final albumin and, specifically, with anantithrombin content greater than or equal to 0.03 mg/g of albumin,giving rise to the present invention.

The albumin solution according to the present invention is obtained bypartial extraction of the antithrombin in a phase of fractionation ofhuman plasma, and, in particular, by chromatographic extraction, forexample, from the plasma, the cryoprecipitate supernatant, the fractionI supernatant or the II+III supernatant.

This partial extraction of the antithrombin may be carried out byinfluencing the parameters that control the chromatographic stage, forexample by varying the chromatography load relationship, so that theeffluent contains sufficient antithrombin to detect a concentrationgreater than or equal to 0.03 mg of active antithrombin/g of albumin inthe final albumin.

In a preferred embodiment, only a portion of the total volume that willyield the final batch of albumin is subjected to chromatographicextraction, the materials that have been subjected to extractionsubsequently being mixed with those that have not been extracted. Thismay be achieved by mixing plasmas, supernatants (of cryoprecipitate, FrIor FrII+III) or fractions (FrIV or FrV) from which the antithrombin hasbeen extracted with others that have not been subjected to saidextraction. This mixture should be in a proportion that is sufficient todetect an antithrombin concentration greater than or equal to 0.03 mg ofantithrombin/g of albumin in the final albumin.

A practical example of an albumin solution according to the invention isgiven hereinafter merely as an example.

EXAMPLE 1

Albumin was prepared from FrV, and the antithrombin was extracted fromthe FrII+III supernatant by heparin-agarose affinity chromatography.

Table 1 shows the antithrombin content of the albumin (final product inconcentration of 20%) as a function of the percentage (%) of the volumeof FrII+III supernatant from which the antithrombin has been extractedby chromatography.

TABLE 1 Extraction of antithrombin in Antithrombin content Antithrombincontent supernatant fractions (mg/ml) in final (mg/g of albumin) inFII + FIII % product (20% alb.) final product 0 (n = 5) 0.020 0.1 50 (n= 2) 0.013 0.065 80 (n = 5) 0.0078 0.039 100 (n = 6) <0.006 <0.03

The antithrombin was extracted in supernatant fractions of 0, 50, 80 and100% respectively while mixing with the corresponding portions ofFII+FIII without antithrombin extraction.

It has been found that, when extracting 100% of antithrombin from theFII+III supernatant, no antithrombin (value lower than the limit ofdetection of the method) was detected in the albumin (final product). Ithas also been found that, when extracting 80% of antithrombin from theFrII+III supernatant and mixing with the remaining 20% (from which theantithrombin has not been extracted), 0.0078 mg of antithrombin per mlof 20% albumin solution are detected.

Table 2 shows the development over time at 5° C. of PKA activity (IU) in20% albumin solutions (final product) in relation to the percentage ofextraction of antithrombin achieved in the FrII+III supernatant.

TABLE 2 Extraction of anti- thrombin Months process (%) 0 1 2 3 5 6 8 912 1 0 <2.0 <2.0 <2.0 — 2.4 — <2.0 — <2.0 2 0 <2.0 <2.0 <2.0 — <2.0 —<2.0 — <2.0 3 50 <2.0 <2.0 <2.0 — <2.0 — <2.0 — <2.0 4 50 <2.0 <2.0 <2.0— <2.0 — <2.0 — 2.5 5 80 <2.0 <2.0 — 2.9 3.0 — — 3.2 5.7 6 80 2.2 3.03.5 4.4 — 2.3 — 6.0 5.6 7 100 8.9 18.5 — 18.2 15.4 — — 22.3 24.2 8 10010.6 23.3 — 24.9 24.0 — — 32.1 32.8

It has been found that in the processes in which 100% of antithrombinwas extracted from the FII+III supernatant, the PKA activity level ishigher from the beginning in the albumin (final product) and, increasesso as to approach the limit set by the European Pharmacopoeia overtwelve months. Conversely, in the processes involving controlled orpartial extraction of the antithrombin, the PKA activity level in thealbumin solution remains at low or undetectable levels.

The description serves merely as an example and does not limit the scopeof the invention, which will merely be defined by the appended claims,with due consideration of equivalents and variations that may beimplemented by experts in the art with knowledge of the presentinvention and that are also included within the scope thereof.

1. A process for reducing prekallikrein activator (PKA) activity inpurified albumin solutions comprising: (a) fractionating human plasma;(b) extracting, by chromatography, a portion of antithrombin from thehuman plasma; and (c) separating a purified albumin solution from thefractionation of human plasma, wherein the purified albumin solution hasan active antithrombin content of 0.03 to 0.1 mg per g albumin, and aPKA activity of less than 35 IU/ml when measured as a 20% (w/v) albuminsolution on storage at 5° C. at 12 months.
 2. The process according toclaim 1, further comprising a step of extracting, by chromatography, aportion of antithrombin from a cryoprecipitate supernatant obtained fromfractionating the human plasma.
 3. The process according to claim 1,further comprising a step of extracting, by chromatography, a portion ofantithrombin from a fraction I supernatant obtained from fractionatingthe human plasma.
 4. The process according to claim 1, furthercomprising a step of extracting, by chromatography, a portion ofantithrombin from a fraction II+III supernatant obtained fromfractionating the human plasma.
 5. The process according to claim 1,further comprising a step of removing a portion of the antithrombin byaffinity chromatography.
 6. The process according to claim 1, furthercomprising a step of removing a portion of the antithrombin byion-exchange chromatography.
 7. The process of claim 1, furthercomprising the step of combining two or more purified albumin solutionsof differing antithrombin concentration to produce an albumin solutionhaving at least 0.03 mg antithrombin per g of albumin.
 8. The process ofclaim 7, wherein at least one of the combined albumin solutions has notbeen subjected to an antithrombin reduction step.
 9. The process ofclaim 1, wherein the purified albumin solution has a PKA activity of 5.7IU/ml or less when measured as a 20% (w/v) albumin solution on storageat 5° C. at 12 months.
 10. The process of claim 1, wherein the resultingalbumin solution has PKA activity ≦3 IU/ml when measured as a 20% (w/v)albumin solution on storage at 5° C. at 1 month.
 11. The process ofclaim 1, further comprising a step of pooling purified albumin solutionsof differing active antithrombin content.